Selective factor Xa inhibitors

ABSTRACT

Novel compounds, their salts and compositions related thereto having activity against mammalian factor Xa are disclosed. The compounds are useful in vitro or in vivo for preventing or treating coagulation disorders.

This application claims priority under 35 U.S.C. § 119 of provisionalapplication no. 60/036,257 Oct. 11, 1996, which is incorporated hereinby reference.

FIELD OF THE INVENTION

This invention relates to novel heterocyclic compounds which are potentand highly selective inhibitors of factor Xa or factor Xa when assembledin the prothrombinase complex. These compounds show selectivity forfactor Xa versus other proteases of the coagulation (e.g. thrombin,fVIIa, flXa) or the fibrinolytic cascades (e.g. plasminogen activators,plasmin).

BACKGROUND OF THE INVENTION

Blood coagulation protects mammalian species when the integrity of theblood vessel wall is damaged and uncontrolled loss of blood threatenssurvival. Coagulation, resulting in the clotting of blood is animportant component of hemostasis. Under normal hemostaticcircumstances, there is maintained an acute balance of clot formationand clot removal (fibrinolysis). The blood coagulation cascade involvesthe conversion of a variety of inactive enzymes (zymogens) into activeenzymes which ultimately convert the soluble plasma protein fibrinogeninto an insoluble matrix of highly cross-linked fibrin. See Davie, etal., "The Coagulation Cascade: Initiation, Maintenance and Regulation"Biochemistry 30:10363-10370 (1991). Blood platelets which adhere todamaged blood vessels are activated and incorporated into the clot andthus play a major role in the initial formation and stabilization ofhemostatic "plugs". In certain diseases of the cardiovascular system,deviations from normal hemostasis push the balance of clot formation andclot dissolution towards life-threatening thrombus formation whenthrombi occlude blood flow in coronary vessels (myocardial infarctions)or limb and pulmonary veins (venous thrombosis). Although platelets andblood coagulation are both involved in thrombus formation, certaincomponents of the coagulation cascade are primarily responsible for theamplification or acceleration of the processes involved in plateletaggregation and fibrin deposition.

A key enzyme in the coagulation cascade as well as in hemostasis, isthrombin. Thrombin is intimately involved in the process of thrombusformation, but under normal circumstances can also play an anticoagulantrole in hemostasis through its ability to convert protein C intoactivated protein C in a thrombomodulin-dependent manner. Thrombin playsa central role in thrombosis through its ability to catalyze thepenultimate conversion of fibrinogen into fibrin and through its potentplatelet activation activity. Direct or indirect inhibition of thrombinactivity has been the focus of a variety of recent anticoagulantstrategies as reviewed by Claeson "Synthetic Peptides andPeptidomimetics as Substrates and Inhibitors of Thrombin and OtherProteases in the Blood Coagulation System", Blood Coag. Fibrinol.5:411-436 (1994). The major classes of anticoagulants currently used inthe clinic directly or indirectly affect thrombin (i.e. heparins,low-molecular weight heparins and coumarins). Thrombin is generated atthe convergence of the intrinsic and extrinsic coagulation pathways bythe prothrombinase complex. The prothrombinase complex is formed whenactivated Factor X (factor Xa) and its non-enzymatic cofactor, factor Vaassemble on phospholipid surfaces in a Ca⁺² -dependent fashion asreviewed by Mann, et al., "Surface-Dependent Reactions of the VitaminK-Dependent Enzymes", Blood 76:1-16 (1990). The prothrombinase complexconverts the zymogen prothrombin into the active procoagulant thrombin.

The location of the prothrombinase complex at the convergence of theintrinsic and extrinsic coagulation pathways, and the significantamplification of thrombin generation (393,000-fold over uncomplexedfactor Xa) mediated by the complex at a limited number of targetedcatalytic units present at vascular lesion sites, suggests thatinhibition of thrombin generation is an ideal method to blockuncontrolled procoagulant activity. Unlike thrombin, which acts on avariety of protein substrates as well as at a specific receptor, factorXa appears to have a single physiologic substrate, namely prothrombin.

Plasma contains an endogenous inhibitor of both the factor VIIa-tissuefactor (TF) complex and factor Xa called tissue factor pathway inhibitor(TFPI). TFPI is a Kunitz-type protease inhibitor with three tandemKunitz domains. TFPI inhibits the TF/fVIIa complex in a two-stepmechanism which includes the initial interaction of the second Kunitzdomain of TFPI with the active site of factor Xa, thereby inhibiting theproteolytic activity of factor Xa. The second step involves theinhibition of the TF/fVIIa complex by formation of a quaternary complexTF/fVIIa/TFPI/fXa as described by Girard, et al., "FunctionalSignificance of the Kunitz-type Inhibitory Domains ofLipoprotein-associated Coagulation Inhibitor", Nature 338:518-520(1989).

Polypeptides derived from hematophagous organisms have been reportedwhich are highly potent and specific inhibitors of factor Xa. U.S. Pat.No. 4,588,587 awarded to Gasic, describes anticoagulant activity in thesaliva of the Mexican leech, Haementeria officinalis. A principalcomponent of this saliva is shown to be the polypeptide factor Xainhibitor, antistasin, by Nutt, et al., "The Amino Acid Sequence ofAntistasin, a Potent Inhibitor of Factor Xa Reveals a Repeated InternalStructure", J. Biol. Chem. 263:10162-10167 (1988).

Another potent and highly specific inhibitor of Factor Xa, tickanticoagulant peptide, has been isolated from the whole body extract ofthe soft tick Ornithidoros moubata, as reported by Waxman, et al., "TickAnticoagulant Peptide (TAP) is a Novel Inhibitor of Blood CoagulationFactor Xa", Science 248:593-596 (1990).

Other polypeptide type inhibitors of factor Xa have been reportedincluding the following citations by: Condra, et al., "Isolation andStructural Characterization of a Potent Inhibitor of Coagulation FactorXa from the Leech Haementeria ghilianii", Thromb. Haemost. 61:437-441(1989); Blankenship, et al., "Amino Acid Sequence of Ghilanten:Anti-coagulant-antimetastatic Principle of the South American Leech,Haementeria ghilianii", Biochem. Biophys. Res. Commun. 166:1384-1389(1990); Brankamp, et al., "Ghilantens: Anticoagulants, AntimetastaticProteins from the South American Leech Haementeria ghilianii", J. Lab.Clin. Med. 115:89-97 (1990); Jacobs, et al., "Isolation andCharacterization of a Coagulation Factor Xa Inhibitor from Black FlySalivary Glands", Thromb. Haemost. 64:235-238 (1990); Rigbi, et al.,"Bovine Factor Xa Inhibiting Factor and Pharmaceutical CompositionsContaining the Same", European Patent Application, 352,903 (1990); Cox,"Coagulation Factor X Inhibitor From the Hundred-pace SnakeDeinagkistrodon acutus venom", Toxicon 31:1445-1457 (1993); Cappello, etal., "Ancylostoma Factor Xa Inhibitor: Partial Purification and itsIdentification as a Major Hookworm-derived Anticoagulant In Vitro", J.Infect. Dis. 167:1474-1477 (1993); Seymour, et al., "Ecotin is a PotentAnticoagulant and Reversible Tight-binding Inhibitor of Factor Xa",Biochemistry 33:3949-3958 (1994).

Factor Xa inhibitory compounds which are not large polypeptide-typeinhibitors have also been reported including: Tidwell, et al.,"Strategies for Anticoagulation With Synthetic Protease Inhibitors. XaInhibitors Versus Thrombin Inhibitors", Thromb. Res. 19:339-349 (1980);Turner, et al., "p-Amidino Esters as Irreversible Inhibitors of FactorIXa and Xa and Thrombin", Biochemistry 25:4929-4935 (1986); Hitomi, etal., "Inhibitory Effect of New Synthetic Protease Inhibitor (FUT-175) onthe Coagulation System", Haemostasis 15:164-168 (1985); Sturzebecher, etal., "Synthetic Inhibitors of Bovine Factor Xa and Thrombin. Comparisonof Their Anticoagulant Efficiency", Thromb. Res. 54:245-252 (1989); Kam,et al., "Mechanism Based Isocoumarin Inhibitors for Trypsin and BloodCoagulation Serine Proteases: New Anticoagulants". Biochemistry27:2547-2557 (1988); Hauptmann, et al., "Comparison of the Anticoagulantand Antithrombotic Effects of Synthetic Thrombin and Factor XaInhibitors", Thromb. Haemost. 63:220-223 (1990); Miyadera, et al,Japanese Patent Application JP 6327488 (1994); Nagahara, et al.,"Dibasic (Amidinoaryl)propanoic Acid Derivatives as Novel BloodCoagulation Factor Xa Inhibitors", J. Med. Chem. 37:1200-1207 (1994);Vlasuk, et al., "Inhibitors of Thrombosis" European Patent Application,WO 93/15756 (1993); and Brunck, et al., "Novel Inhibitors of Factor Xa",European Patent Application, WO 94/13693 (1994). Al-obeidi, et al.,"Factor Xa Inhibitors", WO patent 95/29189, discloses pentapeptideX1-Y-I-R-X2 derivatives as factor Xa inhibitors. Said compounds areuseful for inhibiting blood clotting in the treatment of thrombosis,stroke, and myocardial infarction.

WO 96/18644 to Tamura, et al., describes aromatic heterocyclic thrombininhibitors. WO 95/35313 to Semple, et al., pertains to3-amino-2-oxo-1-piperidineacetic derivative thrombin inhibitors. EP0,512,831 and U.S. Pat. No. 5,281,585, both to Duggan, et al., describefibrinogen receptor antagonists.

SUMMARY OF THE INVENTION

The present invention relates to novel peptide and peptide mimeticanalogs, their pharmaceutically acceptable isomers, salts, hydrates,solvates and prodrug derivatives.

In another aspect, the present invention includes pharmaceuticalcompositions comprising a pharmaceutically effective amount of thecompounds of this invention and a pharmaceutically acceptable carrier.These compositions are useful as potent and specific inhibitors of bloodcoagulation in mammals.

In yet another aspect, the invention relates to methods of using theseinhibitors as therapeutic agents for disease states in mammals whichhave disorders of coagulation such as in the treatment or prevention ofunstable angina, refractory angina, myocardial infarction, transientischemic attacks, thrombotic stroke, embolic stroke, disseminatedintravascular coagulation including the treatment of septic shock, deepvenous thrombosis in the prevention of pulmonary embolism or thetreatment of reocclusion or restenosis of reperfused coronary arteries.These compositions may optionally include anticoagulants, antiplateletagents, and thrombolytic agents.

In other aspects of the invention compounds are provided which areuseful as diagnostic reagents.

In preferred embodiments, the present invention provides compounds ofgeneral formula I: ##STR1## Wherein: R² is H, C₁₋₆ alkyl, C₃₋₆cycloalkyl, C₁₋₃ alkylaryl, C₁₋₃ alkyl-C₃₋₈ cycloalkyl or aryl and R³ isH, C₁₋₆ alkyl, or R² and R³ are taken together to form a carbocyclicring;

R¹³ is H, C₁₋₃ alkyl, C₁₋₃ alkylaryl, aryl, heteroaryl or --CF₃ ;

m is an integer from 0-3;

n is an integer from 0-6;

p is an integer from 0-4;

s is an integer from 0-2;

X is N or CR₁ ; where R₁ is H, C₁₋₃ alkyl, C₁₋₃ alkylaryl, aryl,heteroaryl or --CF₃ ;

Z is N or CR⁴ ; where R¹⁴ is H, C₁₋₃ alkyl, C₁₋₃ alkylaryl, aryl,heteroaryl or --CF₃ ;

A is selected from the group consisting of: a five to ten memberedheterocyclic ring system containing 1-4 heteroatoms selected from thegroup consisting of N, O and S; R⁴ ; --NR⁴ R⁵ ; ##STR2## where R⁴, R⁵,R¹⁷ and R¹⁸ are independently selected from the group consisting of H,--OH, C₁₋₆ alkyl, aryl and C₁₋₄ alkylaryl; R¹⁹ is selected from thegroup consisting of H, --OH, C₁₋₆ alkyl, aryl and C₁₋₄ alkylaryl, or canbe taken together with R¹⁷ or R¹⁸ to form a 5-6 membered ring; and R²⁰is selected from the group consisting of H, --OH, C₁₋₆ alkyl, aryl andC₁₋₄ alkylaryl, or can be taken together with R¹⁸ to form a 5-6 memberedring;

W is C₁₋₆ alkyl, C₃₋₈ cycloalkyl, C₁₋₆ alkenyl, aryl, or a five to tenmembered heterocyclic ring system containing 1-4 heteroatoms selectedfrom the group consisting of N, O and S;

K is selected from the group consisting of a direct link, C₃₋₈cycloalkyl, aryl, or a five to ten membered heterocyclic ring systemcontaining 1-4 heteroatoms selected from the group consisting of N, Oand S;

E is selected from the group consisting of R²⁶, --NR²⁶ R²⁷, ##STR3##where R²⁶, R²⁷, R²⁸ and R²⁹ are independently selected from the groupconsisting of H, --OH, C₁₋₆ alkyl, aryl and C₁₋₄ alkylaryl; R³⁰ isselected from the group consisting of H, C₁₋₆ alkyl, aryl and C₁₋₄alkylaryl, or can be taken together with R²⁸ or R²⁹ to form a 5-6membered ring; and R³¹ is selected from the group consisting of H, C₁₋₆alkyl, aryl and C₁₋₄ alkylaryl, or can be taken together with R²⁹ toform a 5-6 membered ring; with the proviso that when E is R²⁶, then Kmust contain at least one N atom;

Y is selected from the group consisting of H, ##STR4## where R¹⁵ and R¹⁶are independently selected from the group consisting of H, C₁₋₃ alkyland aryl; and G is H, COOR¹¹, CONR¹¹ R¹², --CF₂ CF₃, or a group havingthe formula: ##STR5## where: R²³ is selected from the group consistingof H, C₁₋₆ alkyl, C₂₋₆ alkenyl, C₀₋₆ alkylaryl, C₂₋₆ alkenylaryl, C₀₋₆alkylheterocyclo, C₂₋₆ alkenylheterocyclo, --CF₃ and --CF₂ CF₃ ;

J is --S--, --SO--, --SO₂ --, --O-- or --NR⁶ --, where R⁶ is H, C₁₋₆alkyl or benzyl; and

L is selected from the group consisting of: ##STR6## a C₆₋₁₀heterocyclic ring system substituted by R⁹ and R¹⁰ and containing 1-4heteroatoms selected from N, S and O; where r is an integer from 0-2; R⁷and R⁸ are independently selected from the group consisting of H, C₁₋₆alkyl, aryl, C₁₋₆ alkylaryl, --COOR¹¹, --CONR¹¹ R¹², --CN and --CF₃ ; R⁹and R¹⁰ are independently selected from the group consisting of H, C₁₋₆alkyl, aryl, C₁₋₆ alkylaryl, C₁₋₄ alkyloxy, halogen, --NO₂, --NR¹¹ R¹²,--NR¹¹ COR¹², --O--R¹¹, --O--COR¹¹, --COOR¹¹, --CONR¹¹ R¹², --CN, --CF₃,--SO₂ NR¹¹ R¹² and C₁₋₆ alkyl--O--R₁₁ ; and R¹¹ and R¹² areindependently selected from the group consisting of H, C₁₋₆ alkyl, C₁₋₃alkylaryl and aryl;

U is --O--, --S--, --N-- or --N(H)--; and

V is --O--, --S--, --N-- or --N(H)--; with the proviso that at least oneof U or V is --N-- or --N(H)--;

and all optical isomers thereof.

DETAILED DESCRIPTION OF THE INVENTION Definitions

In accordance with the present invention and as used herein, thefollowing terms are defined with the following meanings, unlessexplicitly stated otherwise.

The term "alkyl" refers to saturated aliphatic groups includingstraight-chain, branched-chain, cyclic groups, and combinations thereof,having the number of carbon atoms specified, or if no number isspecified, having up to 12 carbon atoms. The term "cycloalkyl" refers toa mono-, bi-, or tricyclic aliphatic ring having 3 to 12 carbon atoms,preferably 3 to 7 carbon atoms.

The term "alkenyl" refers to unsaturated aliphatic groups includingstraight-chain, branched-chain, cyclic groups, and combinations thereof,having at least one double bond and having the number of carbon atomsspecified.

The term "aryl" refers to an unsubstituted or substituted aromaticring(s), substituted with one, two or three substituents such as, by wayof example and not limitation, C₁₋₆ alkoxy, C₁₋₆ alkyl, C₁₋₆ alkylamino,hydroxy, halogen, cyano (--CN), hydroxyl, mercapto, nitro (--NO₂),thioalkoxy, carboxaldehyde, carboxyl, carboalkoxy, carboxamide, --NR'R",--NR'COR", --OR, --OCOR, --COOR, --CONR'R", --CF₃, --SO₂ NR'R" and C₁₋₆alkyl--OR; aryl, C₁₋₆ alkylaryl (where the R groups can be H, C₁₋₆alkyl, C₁₋₃ alkylaryl and aryl), including but not limited tocarbocyclic aryl, heterocyclic aryl, biaryl and triaryl groups and thelike, all of which may be optionally substituted. Preferred aryl groupsinclude phenyl, halophenyl, C₁₋₆ alkylphenyl, naphthyl, biphenyl,phenanthrenyl, naphthacenyl, and aromatic heterocyclics or heteroaryls,the latter of which is an aryl group containing one to four heteroatomsselected from the group consisting of nitrogen, oxygen and sulfur. Arylgroups preferably have 5-14 carbon atoms making up the ring(s)structure, while heteroaryls preferably have 1-4 heteroatoms, with theremaining 4-10 atoms being carbon atoms.

The terms "heterocyclo" and "hetero cyclic ring system" as used hereinrefer to any saturated or unsaturated mono- or bicyclic ring system,containing from one to four heteroatoms, selected from the groupconsisting of nitrogen, oxygen and sulfur. A typical heterocyclic ringsystem will have five to ten members, 1-4 of which are heteroatoms.Typical examples of monocyclic ring systems include piperidinyl,pyrrolidinyl, pyridinyl, piperidonyl, pyrrolidonyl and thiazolyl, whileexamples of bicyclic ring systems include benzimidazolyl, benzothiazolyland benzoxazolyl, all of which may be substituted.

The term "carbocyclic ring" as used herein refers to any saturated orunsaturated ring containing from three to six carbon atoms.

The terms "alkylaryl" and "alkenylaryl" as used herein refer to an alkylgroup or alkenyl group, respectively, having the number of carbon atomsdesignated, appended to one, two, or three aryl groups. The term benzylas used herein refers to --CH₂ --C₆ H₅.

The term "alkoxy" as used herein refers to an alkyl linked to an oxygenatom, such as methoxy, ethoxy, and so forth.

The terms "halogen" as used herein refer to Cl, Br, F or I substituents.

The term "direct link" as used herein refers to a bond directly linkingthe substituents on each side of the direct link. When two adjacentsubstituents are defined as each being a "direct link", it is consideredto be a single bond.

Two substituents are "taken together to form a 5-6 membered ring" meansthat an ethylene or a propylene bridge, respectively, is formed betweenthe two substituents.

The term "pharmaceutically acceptable salts" includes salts of compoundsderived from the combination of a compound and an organic or inorganicacid. These compounds are useful in both free base and salt form. Inpractice, the use of the salt form amounts to use of the base form; bothacid and base addition salts are within the scope of the presentinvention.

"Pharmaceutically acceptable acid addition salt" refers to those saltswhich retain the biological effectiveness and properties of the freebases and which are not biologically or otherwise undesirable, formedwith inorganic acids such as hydrochloric acid, hydrobromic acid,sulfuric acid, nitric acid, phosphoric acid and the like, and organicacids such as acetic acid, propionic acid, glycolic acid, pyruvic acid,oxalic acid, maleic acid, malonic acid, succinic acid, fumaric acid,tartaric acid, citric acid, benzoic acid, cinnamic acid, mandelic acid,methanesulfonic acid, ethanesulfonic acid, p-toluenesulfonic acid,salicylic acid and the like.

"Pharmaceutically acceptable base addition salts" include those derivedfrom inorganic bases such as sodium, potassium, lithium, ammonium,calcium, magnesium, iron, zinc, copper, manganese, and aluminum bases,and the like. Particularly preferred are the ammonium, potassium,sodium, calcium and magnesium salts. Salts derived from pharmaceuticallyacceptable organic nontoxic bases include salts of primary, secondary,and tertiary amines, substituted amines including naturally occurringsubstituted amines, cyclic amines and basic ion exchange resins, such asisopropylamine, trimethylamine, diethylamine, triethylamine,tripropylamine, ethanolamine, 2-diethylaminoethanol, trimethamine,dicyclohexylamine, lysine, arginine, histidine, caffeine, procaine,hydrabaminie, choline, betaine, ethylenediamine, glucosamine,methylglucamine, theobromine, purines, piperizine, piperidine,N-ethylpiperidine, polyamine resins and the like. Particularly preferredorganic nontoxic bases are isopropylamine, diethylamine, ethanolamine,trimethamine, dicyclohexylamine, choline, and caffeine.

"Biological property" for the purposes herein means an in vivo effectoror antigenic function or activity that is directly or indirectlyperformed by a compound of this invention. Effector functions includereceptor or ligand binding, any enzyme activity or enzyme modulatoryactivity, any carrier binding activity, any hormonal activity, anyactivity in promoting or inhibiting adhesion of cells to anextracellular matrix or cell surface molecules, or any structural role.Antigenic functions include possession of an epitope or antigenic sitethat is capable of reacting with antibodies raised against it. Thebiological properties of the compounds of the present invention can bereadily characterized by the methods described in Examples 7 and 8 andby such other methods as are well known in the art.

In addition, the following abbreviations are used in this application:

"Boc" refers to t-butoxycarbonyl.

"BOP" refers to benzotriazol-1-yloxy-tris-(dimethylamino) phosphoniumhexafluorophosphate.

"DIEA" refers to diisopropylethylamine.

"DMF" refers to N,N-dimethylformamide.

"Et₂ O" refers to diethyl ether.

"EtOAc" refers to ethyl acetate.

"HF" refers to hydrogen fluoride.

"ICH₂ COOEt" refers to ethyl iodoacetate.

"LiN(TMS)₂ " refers to lithium bis-trimethyl silyl amide.

"MeSEt" refers to methyl ethyl sulfide.

"TFA" refers to trifluoroacetic acid.

"THF" refers to tetrahydrofuran.

"TMSI" refers to trimethyl silyl iodide.

"Tos" refers to p-toluenesulfonyl.

In the compounds of this invention, carbon atoms bonded to fournon-identical substituents are asymmetric. Accordingly, the compoundsmay exist as diastereoisomers, enantiomers or mixtures thereof. Thesyntheses described herein may employ racemates, enantiomers ordiastereomers as starting materials or intermediates. Diastereomericproducts resulting from such syntheses may be separated bychromatographic or crystallization methods, or by other methods known inthe art. Likewise, enantiomeric product mixtures may be separated usingthe same techniques or by other methods known in the art. Each of theasymmetric carbon atoms, when present in the compounds of thisinvention, may be in one of two configurations (R or S) and both arewithin the scope of the present invention. In the processes describedabove., the final products may, in some cases, contain a small amount ofdiastereomeric or enantiomeric products; however, these products do notaffect their therapeutic or diagnostic application.

In all of the peptides of the invention, one or more amide linkages(--CO--NH--) may optionally be replaced with another linkage which is anisostere such as --CH₂ NH--, --CH₂ S--, --CH₂ --O--, --CH₂ CH₂ --,--CH═CH--(cis and trans), --COCH₂ --, --CH(OH)CH₂ --, --CH₂ SO--, and--CH₂ SO₂ --. This replacement can be made by methods known in the art.The following references describe preparation of peptide analogs whichinclude these alternative-linking moieties: Spatola, "Peptide BackboneModifications" (general review) Vega Data Vol. 1, Issue 3, (March 1983);Spatola, "Chemistry and Biochemistry of Anino Acids, Peptides andProteins," (general review) B. Weinstein, eds., Marcel Dekker, New York,p. 267 (1983); Morley, Trends Pharm. Sci. (general review) pp. 463-468(1980); Hudson, et al., Int. J. Pept. Prot. Res. 14:177-185 (1979)(--CH₂ NH--, --CH₂ CH₂ --); Spatola, et al., Life Sci. 38:1243-1249(1986) (--CH₂ --S); Harn, J. Chem. Soc. Perkin Trans. I pp.307-314(1982) (--CH═CH--, cis and trans); Almquist, et al., J. Med. Chem.23:1392-1398 (1980) (--COCH₂ --); Jennings-White, et al., TetrahedronLett. 23:2533 (--COCH₂ --) (1982); Szelke, et al., European ApplicationEP 45665; CA:97:39405 (1982) (--CH(OH)CH₂ --); Holladay, et al.,Tetrahedron Lett 24:4401-4404 (1983) (--CH(OH)CH₂ --); and Hruby, LifeSci. 31:189-199 (1982) (--CH₂ --S--).

Preferred Embodiments

This invention relates to a new class of peptide derivatives selectedfrom those of general formula I which are potent and specific inhibitorsof Xa, their pharmaceutically acceptable compositions thereof, and themethods of using them as therapeutic agents for disease states inmammals characterized by abnormal thrombosis: ##STR7## Wherein: R² is H,C₁₋₆ alkyl, C₃₋₆ cycloalkyl, C₁₋₃ alkylaryl, C₁₋₃ alkyl-C₃₋₈ cycloalkylor aryl and R³ is H, C₁₋₆ alkyl, or R² and R³ are taken together to forma carbocyclic ring;

R¹³ is H, C₁₋₃ alkyl, C₁₋₃ alkylaryl, aryl, heteroaryl or --CF₃ ;

X is N or CR₁ ; where R₁ is H, C₁₋₃ alkyl, C₁₋₃ alkylaryl, aryl,heteroaryl or --CF₃ ;

Z is N or CR¹⁴ ; where R¹⁴ is H, C₁₋₃ alkyl, C₁₋₃ alkylaryl, aryl,heteroaryl or --CF₃ ;

m is an integer from 0-3;

n is an integer from 0-6;

p is an integer from 0-4;

s is an integer from 0-2;

A is selected from the group consisting of: a five to ten memberedheterocyclic ring system containing 1-4 heteroatoms selected from thegroup consisting of N, O and S; R⁴ ; --NR⁴ R⁵ ; ##STR8## where R⁴, R⁵,R¹⁷ and R¹⁸ are independently selected from the group consisting of H,--OH, C₁₋₆ alkyl, aryl and C₁₋₄ alkylaryl; R¹⁹ is selected from thegroup consisting of H, --OH, C₁₋₆ alkyl, aryl and C₁₋₄ alkylaryl, or canbe taken together with R¹⁷ or R¹⁸ to form a 5-6 membered ring; and R²⁰is selected from the group consisting of H, --OH, C₁₋₆ alkyl, aryl andC₁₋₄ alkylaryl, or can be taken together with R¹⁸ to form a 5-6 memberedring;

W is C₁₋₆ alkyl, C₃₋₈ cycloalkyl, C₁₋₆ alkenyl, aryl, or a five to tenmembered heterocyclic ring system containing 1-4 heteroatoms selectedfrom the group consisting of N, O and S;

K is selected from the group consisting of a direct link, C₃₋₈cycloalkyl, aryl, or a five to ten membered heterocyclic ring systemcontaining 1-4 heteroatoms selected from the group consisting of N, Oand S;

E is selected from the group consisting of R²⁶, --NR²⁶ R²⁷, ##STR9##where R²⁶, R²⁷, R²⁸ and R²⁹ are independently selected from the groupconsisting of H, --OH, C₁₋₆ alkyl, aryl and C₁₋₄ alkylaryl; R³⁰ isselected from the group consisting of H, C₁₋₆ alkyl, aryl and C₁₋₄alkylaryl, or can be taken together with R²⁸ or R²⁹ to form a 5-6membered ring; and R³¹ is selected from the group consisting of H, C₁₋₆alkyl, aryl and C₁₋₄ alkylaryl, or can be taken together with R²⁹ toform a 5-6 membered ring; with the proviso that when E is R²⁶, then Kmust contain at least one N atom;

Y is selected from the group consisting of H, ##STR10## where R¹⁵ andR¹⁶ are independently selected from the group consisting of H, C₁₋₃alkyl and aryl; and G is H, COOR¹¹, CONR¹¹ R¹², --CF₃, --CF₂ CF₃ or agroup having the formula: ##STR11## where: R²³ is selected from thegroup consisting of H, C₁₋₆ alkyl, C₂₋₆ alkenyl, C₀₋₆ akylaryl, C₂₋₆alkenylaryl, C₀₋₆ alkylheterocyclo, C₂₋₆ alkenylheterocyclo, --CF₃ and--CF₂ CF₃ ;

J is --S--, --SO--, --SO₂ --, --O-- or --NR⁶ --, where R⁶ is H, C₁₋₆alkyl or benzyl; and

L is selected from the group consisting of: ##STR12## a C₆₋₁₀heterocyclic ring system substituted by R⁹ and R¹⁰ and containing 1-4heteroatoms selected from N, S and O; where r is an integer from 0-2; R⁷and R⁸ are independently selected from the group consisting of H, C₁₋₆alkyl, aryl, C₁₋₆ alkylaryl, --COOR¹¹, --CONR¹¹ R¹², --CN and --CF₃ ; R⁹and R¹⁰ are independently selected from the group consisting of H, C₁₋₆alkyl, aryl, C₁₋₆ alkylaryl, C₁₋₄ alkyloxy, halogen, --NO₂, --NR¹¹ R¹²,--NR¹¹ COR¹², --O--R¹¹, --O--COR¹¹, --COOR¹¹, --CONR¹¹ R¹², --CN, --CF₃,--SO₂ NR¹¹ R¹² and C₁₋₆ alkyl--O--R¹¹ ; and R¹¹ and R¹² areindependently selected from the group consisting of H, C₁₋₆ alkyl, C₁₋₃alkylaryl and aryl;

U is --O--, --S--, --N-- or --N(H)--; and

V is --O--, --S--, --N-- or --N(H)--; with the proviso that at least oneof U or V is --N-- or --N(H)--;

and all optical isomers thereof.

Preferred R₂ substituents are H and C₁₋₆ alkyl; more preferably H.

R³ is preferably H.

R¹³ is preferably H, C₁₋₃ alkyl or --CF₃ ; more preferably H.

Preferably at least one of "X" or "Z" is CR₁ or CR¹⁴, respectively.

Preferred R₁ substituents are H, C₁₋₃ alkyl and --CF₃ ; more preferablyH.

R¹⁴ is preferably H, C₁₋₃ alkyl or --CF₃ ; more preferably H.

The integer "m" is preferably from 0-1; more preferably 0.

The integer "n" is preferably from 1-4.

The integer "p" is preferably 3.

The integer "s" is preferably 0.

Preferred "A" substituents are R⁴, --NR⁴ R⁵, ##STR13##

R⁴ is preferably H, --OH or C₁₋₆ alkyl; more preferably H, --OH ormethyl.

R⁵ is preferably H, --OH or C₁₋₆ alkyl; more preferably H, --OH ormethyl.

Preferred "W" substituents are C₁₋₄ alkyl, C₅₋₆ cycloalkyl, aryl, or afive to ten membered heterocyclic ring system containing at least one Nheteroatom; more preferably C₁₋₄ alkyl, aryl, or a five to ten memberedheterocyclic ring system containing at least one N heteroatom.

K is preferably a direct link.

In the "E" substituent, it is preferred that R²⁶, R²⁷, R²⁸, R²⁹, R³⁰ andR³¹ are independently selected from the group consisting of H and C₁₋₆alkyl, more preferably H and methyl. Particularly preferred "E"substituents are --NH₂, --NHC(═NH)--NH₂ and --SC(═NH)--NH₂ ; morepreferably --NHC(═NH)--NH₂ and --SC(═NH)--NH₂.

Preferred "Y" substituents are: ##STR14##

R¹⁵ is preferably H.

R¹⁶ is preferably H.

The "G" substituent is preferably a group having the formula: ##STR15##

The "J" substituent is preferably --S--, --O-- or --NR⁶ --.

R⁶ is preferably H.

The "L" substituent is preferably: ##STR16## more preferably: ##STR17##

R⁷ is preferably H.

R⁸ is preferably H.

R⁹ is preferably H, --O--R¹¹, --COOR¹¹, --CONR¹¹ R¹² or --CF₃ ; morepreferably H.

R¹⁰ is preferably H, --O--R¹¹, --COOR¹¹, --CONR¹¹ R¹² or --CF₃ ; morepreferably H.

R¹¹ is preferably H.

R¹² is preferably H.

In one embodiment of the invention R², R³ and R¹³ are H, Z is CH, p=3,s=0, K is a direct link, E is --NHC(═NH)--NH₂ and Y is --CO-G, where Gis a group having the formula: ##STR18## where J and L are as definedabove. This is also illustrated as a preferred group of compoundsdefined by the general structural formula II as: ##STR19## wherein: X isN or CR₁ ; where R₁ is H, C₁₋₃ alkyl, C₁₋₃ alkylaryl, aryl, heteroarylor --CF₃ ;

m is an integer from 0-3;

n is an integer from 0-6;

A is selected from the group consisting of R⁴, --NR⁴ R⁵, ##STR20## whereR⁴, R⁵, R¹⁷ and R¹⁸ are independently selected from the group consistingof H, --OH, C₁₋₆ alkyl, aryl and C₁₋₄ alkylaryl; R¹⁹ is selected fromthe group consisting of H, --OH, C₁₋₆ alkyl, aryl and C₁₋₄ alkylaryl, orcan be taken together with R¹⁷ or R¹⁸ to form a 5-6 membered ring; andR²⁰ is selected from the group consisting of H, --OH, C₁₋₆ alkyl, aryland C₁₋₄ alkyaryl, or can be taken together with R¹⁸ to form a 5-6membered ring;

W is C₁₋₆ alkyl, C₃₋₈ cycloalkyl, C₁₋₆ alkenyl, aryl, or a five to tenmembered heterocyclic ring system containing 1-4 heteroatoms selectedfrom the group consisting of N, O and S; with the proviso that when A isR⁴, then W must contain at least one N atom;

J is --S--, --SO--, --SO₂ --, --O-- or --NR⁶ --, where R⁶ is H; and

L is selected from the group consisting of: ##STR21## a C₆₋₁₀heterocyclic ring system substituted by R⁹ and R¹⁰ and containing 1-4heteroatoms selected from N, S and O; where r is an integer from 0-1; R⁷and R⁸ are independently selected from the group consisting of H, C₁₋₆alkyl, aryl, C₁₋₆ alkylaryl, --COOR¹¹, --CONR¹¹ R¹², --CN and --CF₃ ; R⁹and R¹⁰ are independently selected from the group consisting of H, C₁₋₆alkyl, aryl, C₁₋₆ alkylaryl, C₁₋₄ alkyloxy, halogen, --NO₂, --NR¹¹ R¹²,--NR¹¹ COR¹², --O--R¹¹, --O--COR¹¹, --COOR¹¹, --CONR¹¹ R¹², --CN, --CF₃,--SO₂ NR¹¹ R¹² and C₁₋₆ alkyl-O--R¹¹ ; and R¹¹ and R¹² are independentlyselected from the group consisting of H, C₁₋₆ alkyl, C₁₋₃ alkylaryl andaryl; and all optical isomers thereof.

A preferred embodiment of compounds of general formula II have thefollowing stereochemistry: ##STR22##

In yet another embodiment of the invention, R², R³ and R¹³ are H, X andZ are CH, p=3,s=0, K is a direct link, E is --NHC(═NH)--NH₂, and Y is--CO--G, where G is a group having the formula: ##STR23## where J is--S--and L is the group: ##STR24## where R⁹ and R¹⁰ are H. This is alsoillustrated as a preferred group of compounds defined by the generalstructural formula III as: ##STR25## wherein: m is an integer from 0-3;

n is an integer from 0-6;

A is selected from the group consisting of R⁴, --NR⁴ R⁵, ##STR26## whereR⁴, R⁵, R¹⁷ and R¹⁸ are independently selected from the group consistingof H, --OH, C₁₋₆ alkyl, aryl and C₁₋₄ alkylaryl; R¹⁹ is selected fromthe group consisting of H, --OH, C₁₋₆ alkyl, aryl and C₁₋₄ alkylaryl, orcan be taken together with R¹⁷ or R¹⁸ to form a 5-6 membered ring; andR²⁰ is selected from the group consisting of H, --OH, C₁₋₆ alkyl, aryland C₁₋₄ alkylaryl, or can be taken together with R¹⁸ to form a 5-6membered ring;

W is C₁₋₆ alkyl, C₃₋₈ cycloalkyl, C₁₋₆ alkenyl, aryl, or a five to tenmembered heterocyclic ring system containing 1-4 heteroatoms selectedfrom the group consisting of N, O and S; with the proviso that when A isR⁴, then W must contain at least one N atom;

and all optical isomers thereof.

A preferred embodiment of compounds of general formula III have thefollowing stereochemistry: ##STR27##

This invention also encompasses all pharmaceutically acceptable isomers,salts, hydrates and solvates of the compounds of formulas I, II and III.In addition, the compounds of formulas I, II and III can exist invarious isomeric and tautomeric forms, and all such forms are meant tobe included in the invention, along with pharmaceutically acceptablesalts, hydrates and solvates of such isomers and tautomers.

The compounds of this invention may be isolated as the free acid or baseor converted to salts of various inorganic and organic acids and bases.Such salts are within the scope of this invention. Non-toxic andphysiologically compatible salts are particularly useful although otherless desirable salts may have use in the processes of isolation andpurification.

A number of methods are useful for the preparation of the saltsdescribed above and are known to those skilled in the art. For example,the free acid or free base form of a compound of one of the formulasabove can be reacted with one or more molar equivalents of the desiredacid or base in a solvent or solvent mixture in which the salt isinsoluble, or in a solvent like water after which the solvent is removedby evaporation, distillation or freeze drying. Alternatively, the freeacid or base form of the product may be passed over an ion exchangeresin to form the desired salt or one salt form of the product may beconverted to another using the same general process.

This invention also encompasses prodrug derivatives of the compoundscontained herein. The term "prodrug" refers to a pharmacologicallyinactive derivative of a parent drug molecule that requiresbiotransformation, either spontaneous or enzymatic, within the organismto release the active drug. Prodrugs are variations or derivatives ofthe compounds of this invention which have groups cleavable undermetabolic conditions. Prodrugs become the compounds of the inventionwhich are pharmaceutically active in vivo, when they undergo solvolysisunder physiological conditions or undergo enzymatic degradation. Prodrugcompounds of this invention may be called single, double, triple etc.,depending on the number of biotransformation steps required to releasethe active drug within the organism, and indicating the number offunctionalities present in a precursor-type form. Prodrug forms oftenoffer advantages of solubility, tissue compatibility, or delayed releasein the mammalian organism (see, Bundgard, Design of Prodrugs, pp.7-9,21-24, Elsevier, Amsterdam 1985 and Silverman, The Organic Chemistryof Drug Design and Drug Action, pp. 352-401, Academic Press, San Diego,Calif., 1992). Prodrugs commonly known in the art include acidderivatives well known to practitioners of the art, such as, forexample, esters prepared by reaction of the parent acids with a suitablealcohol, or amides prepared by reaction of the parent acid compound withan amine, or basic groups reacted to form an acylated base derivative.Moreover, the prodrug derivatives of this invention may be combined withother features herein taught to enhance bioavailability.

The following structures are illustrative of the compounds of thepresent invention and are not intended to be limiting in any manner:##STR28##

As mentioned above, the compounds of this invention find utility astherapeutic agents for disease states in mammals which have disorders ofcoagulation such as in the treatment or prevention of unstable angina,refractory angina, myocardial infarction, transient ischemic attacks,thrombotic stroke, embolic stroke, disseminated intravascularcoagulation including the treatment of septic shock, deep venousthrombosis in the prevention of pulmonary embolism or the treatment ofreocclusion or restenosis of reperfused coronary arteries. Further,these compounds are useful for the treatment or prophylaxis of thosediseases which involve the production and/or action of factorXa/prothrombinase complex. This includes a number of thrombotic andprothrombotic states in which the coagulation cascade is activated whichinclude but are not limited to, deep venous thrombosis, pulmonaryembolism, myocardial infarction, stroke, thromboembolic complications ofsurgery and peripheral arterial occlusion.

Accordingly, a method for preventing or treating a condition in a mammalcharacterized by undesired thrombosis comprises administering to themammal a therapeutically effective amount of a compound of thisinvention. In addition to the disease states noted above, other diseasestreatable or preventable by the administration of compounds of thisinvention include, without limitation, occlusive coronary thrombusformation resulting from either thrombolytic therapy or percutaneoustransluminal coronary angioplasty, thrombus formation in the venousvasculature, disseminated intravascular coagulopathy, a conditionwherein there is rapid consumption of coagulation factors and systemiccoagulation which results in the formation of life-threatening thrombioccurring throughout the microvasculature leading to widespread organfailure, hemorrhagic stroke, renal dialysis, blood oxygenation, andcardiac catheterization.

The compounds of the invention also find utility in a method forinhibiting the coagulation biological samples, which comprises theadministration of a compound of the invention.

The compounds of the present invention may also be used in combinationwith other therapeutic or diagnostic agents. In certain preferredembodiments, the compounds of this invention may be coadministered alongwith other compounds typically prescribed for these conditions accordingto generally accepted medical practice such as anticoagulant agents,thrombolytic agents, or other antithrombotics, including plateletaggregation inhibitors, tissue plasminogen activators, urokinase,prourokinase, streptokinase, heparin, aspirin, or warfarin. Thecompounds of the present invention may act in a synergistic fashion toprevent reocclusion following a successful thrombolytic therapy and/orreduce the time to reperfusion. These compounds may also allow forreduced doses of the thrombolytic agents to be used and thereforeminimize potential hemorrhagic side-effects. The compounds of thisinvention can be utilized in vivo, ordinarily in mammals such asprimates, (e.g. humans), sheep, horses, cattle, pigs, dogs, cats, ratsand mice, or in vitro.

The biological properties of the compounds of the present invention canbe readily characterized by methods that are well known in the art, forexample by the in vitro protease activity assays and in vivo studies toevaluate antithrombotic efficacy, and effects on hemostasis andhematological parameters, such as are illustrated in the examples.

Diagnostic applications of the compounds of this invention willtypically utilize formulations in the form of solutions or suspensions.In the management of thrombotic disorders the compounds of thisinvention may be utilized in compositions such as tablets, capsules orelixirs for oral administration, suppositories, sterile solutions orsuspensions or injectable administration, and the like, or incorporatedinto shaped articles. Subjects in need of treatment (typicallymammalian) using the compounds of this invention can be administereddosages that will provide optimal efficacy. The dose and method ofadministration will vary from subject to subject and be dependent uponsuch factors as the type of mammal being treated, its sex, weight, diet,concurrent medication, overall clinical condition, the particularcompounds employed, the specific use for which these compounds areemployed, and other factors which those skilled in the medical arts willrecognize.

Formulations of the compounds of this invention are prepared for storageor administration by mixing the compound having a desired degree ofpurity with physiologically acceptable carriers, excipients, stabilizersetc., and may be provided in sustained release or timed releaseformulations. Acceptable carriers or diluents for therapeutic use arewell known in the pharmaceutical field, and are described, for example,in Remington's Pharmaceutical Sciences, Mack Publishing Co., (A. R.Gennaro edit 1985). Such materials are nontoxic to the recipients at thedosages and concentrations employed, and include buffers such asphosphate, citrate, acetate and other organic acid salts, antioxidantssuch as ascorbic acid, low molecular weight (less than about tenresidues) peptides such as polyarginine, proteins, such as serumalbumin, gelatin, or immunoglobulins, hydrophilic polymers such aspolyvinylpyrrolidinone, amino acids such as glycine, glutamic acid,aspartic acid, or arginine, monosaccharides, disaccharides, and othercarbohydrates including cellulose or its derivatives, glucose, mannoseor dextrins, chelating agents such as EDTA, sugar alcohols such asmannitol or sorbitol, counterions such as sodium and/or nonionicsurfactants such as Tween, Pluronics or polyethyleneglycol.

Dosage formulations of the compounds of this invention to be used fortherapeutic administration must be sterile. Sterility is readilyaccomplished by filtration through sterile membranes such as 0.2 micronmembranes, or by other conventional methods. Formulations typically willbe stored in lyophilized form or as an aqueous solution. The pH of thepreparations of this invention typically will be 3-11, more preferably5-9 and most preferably 7-8. It will be understood that use of certainof the foregoing excipients, carriers, or stabilizers will result in theformation of cyclic polypeptide salts. While the preferred route ofadministration is by injection, other methods of administration are alsoanticipated such as orally, intravenously (bolus and/or infusion),subcutaneously, intramuscularly, colonically, rectally, nasally,transdermally or intraperitoneally, employing a variety of dosage formssuch as suppositories, implanted pellets or small cylinders, aerosols,oral dosage formulations and topical formulations such as ointments,drops and dermal patches. The compounds of this invention are desirablyincorporated into shaped articles such as implants which may employinert materials such as biodegradable polymers or synthetic silicones,for example, Silastic, silicone rubber or other polymers commerciallyavailable.

The compounds of the invention may also be administered in the form ofliposome delivery systems, such as small unilamellar vesicles, largeunilamellar vesicles and multilamellar vesicles. Liposomes can be formedfrom a variety of lipids, such as cholesterol, stearylamine orphosphatidylcholines.

The compounds of this invention may also be delivered by the use ofantibodies, antibody fragments, growth factors, hormones, or othertargeting moieties, to which the compound molecules are coupled. Thecompounds of this invention may also be coupled with suitable polymersas targetable drug carriers. Such polymers can includepolyvinylpyrrolidinone, pyran copolymer,polyhydroxy-propyl-methacrylamide-phenol,polyhydroxyethyl-aspartamide-phenol, or polyethyleneoxide-polylysinesubstituted with palmitoyl residues. Furthermore, compounds of theinvention may be coupled to a class of biodegradable polymers useful inachieving controlled release of a drug, for example polylactic acid,polyglycolic acid, copolymers of polylactic and polyglycolic acid,polyepsilon caprolactone, polyhydroxy butyric acid, polyorthoesters,polyacetals, polydihydropyrans, polycyanoacrylates and cross linked oramphipathic block copolymers of hydrogels. Polymers and semipermeablepolymer matrices may be formed into shaped articles, such as valves,stents, tubing, prostheses and the like.

Therapeutic compound liquid formulations generally are placed into acontainer having a sterile access port, for example, an intravenoussolution bag or vial having a stopper pierceable by hypodermic injectionneedle.

Therapeutically effective dosages may be determined by either in vitroor in vivo methods. For each particular compound of the presentinvention, individual determinations may be made to determine theoptimal dosage required. The range of therapeutically effective dosageswill be influenced by the route of administration, the therapeuticobjectives and the condition of the patient. For injection by hypodermicneedle, it may be assumed the dosage is delivered into the body'sfluids. For other routes of administration, the absorption efficiencymust be individually determined for each compound by methods well knownin pharmacology. Accordingly, it may be necessary for the therapist totiter the dosage and modify the route of administration as required toobtain the optimal therapeutic effect. The determination of effectivedosage levels, that is, the dosage levels necessary to achieve thedesired result, will be readily determined by one skilled in the art.Typically, applications of compound are commenced at lower dosagelevels, with dosage levels being increased until the desired effect isachieved.

The compounds of the invention can be administered orally orparenterally in an effective amount within the dosage range of about 0.1to 100 mg/kg, preferably about 0.5 to 50 mg/kg and more preferably about1 to 20 mg/kg on a regimen in a single or 2 to 4 divided daily dosesand/or continuous infusion.

Typically, about 5 to 500 mg of a compound or mixture of compounds ofthis invention, as the free acid or base form or as a pharmaceuticallyacceptable salt, is compounded with a physiologically acceptablevehicle, carrier, excipient, binder, preservative, stabilizer, dye,flavor etc., as called for by accepted pharmaceutical practice. Theamount of active ingredient in these compositions is such that asuitable dosage in the range indicated is obtained.

Typical adjuvants which may be incorporated into tablets, capsules andthe like are binders such as acacia, corn starch or gelatin, andexcipients such as microcrystalline cellulose, disintegrating agentslike corn starch or alginic acid, lubricants such as magnesium stearate,sweetening agents such as sucrose or lactose, or flavoring agents. Whena dosage form is a capsule, in addition to the above materials it mayalso contain liquid carriers such as water, saline, or a fatty oil.Other materials of various types may be used as coatings or as modifiersof the physical form of the dosage unit. Sterile compositions forinjection can be formulated according to conventional pharmaceuticalpractice. For example, dissolution or suspension of the active compoundin a vehicle such as an oil or a synthetic fatty vehicle like ethyloleate, or into a liposome may be desired. Buffers, preservatives,antioxidants and the like can be incorporated according to acceptedpharmaceutical practice.

Preparation of the Disclosed Compounds

The compounds of the present invention may be synthesized by eithersolid or liquid phase methods described and referenced in standardtextbooks, or by a combination of both methods. These methods are wellknown in the art. See, Bodanszky, "The Principles of Peptide Synthesis",Hafner, et al., Eds., Springer-Verlag, Berlin, 1984.

Starting materials used in any of these methods are commerciallyavailable from chemical vendors such as Aldrich, Sigma, NovaBiochemicals, Bachem Biosciences, and the like, or may be readilysynthesized by known procedures.

Reactions are carried out in standard laboratory glassware and reactionvessels under reaction conditions of standard temperature and pressure,except where otherwise indicated.

During the synthesis of these compounds, the functional groups of theamino acid derivatives used in these methods are protected by blockinggroups to prevent cross reaction during the coupling procedure. Examplesof suitable blocking groups and their use are described in "ThePeptides: Analysis, Synthesis, Biology", Academic Press, Vol. 3 (Gross,et al., Eds., 1981) and Vol. 9 (1987), the disclosures of which areincorporated herein by reference.

One exemplary synthesis scheme is outlined directly below, and thespecific steps are described in the Examples. The reaction products areisolated and purified by conventional methods, typically by solventextraction into a compatible solvent. The products may be furtherpurified by column chromatography or other appropriate methods.##STR29## Most compounds are purified by reversed-phase HPLC andcharacterized by ion-spray MS spectrometry. ##STR30##

The chemical reactions described in Scheme I can easily be modified andcombined with other techniques that are well known in the art to produceother compounds within the family of formula I.

Methods of incorporating "A" substituents such as R⁴, --NR⁴ R⁵,##STR31## are well known in the art. The chemistry of using the variousR⁴ and R⁵ substituents (H, C₁₋₆ alkyl and C₁₋₄ alkylaryl) is also wellknown in the art. Methods of incorporating "W" substituents such as C₁₋₆alkyl, C₃₋₈ cycloalkyl, aryl, or a five to ten membered heterocyclicring system containing 1-4 heteroatoms selected from the groupconsisting of N, O and S, are also well known in the art. In addition, Aand W substituents are illustrated in Example 6.

The "X" and "Z" substituents illustrated in Fragment-1 are --CR₁ andCR¹⁴, respectively, where R₁ and R¹⁴ are H. Fragments similar to thestructure of Fragment-2, where "X" and/or "Z" are N or where R₁ and R¹⁴are other suitable substituents such as C₁₋₃ alkyl, C₁₋₃ alkylaryl,aryl, heteroaryl or --CF₃, can easily be synthesized by techniques thatare well known in the art.

The "R₂ " substituent illustrated in Fragment-1 are --H. Fragmentssimilar to the structure of Fragment-1, where R₂ is C₁₋₆ alkyl, C₃₋₆cycloalkyl, C₁₋₃ alkylaryl, or aryl, can easily be synthesized bytechniques that are well known in the art. Similarly, the R¹³substituent illustrated in Fragment-1 is H, but the other R¹³ groups(C₁₋₃ alkyl, C₁₋₃ alkylaryl, aryl, heteroaryl and --CF₃) can besynthesized by techniques that are well known in the art. See, forexample, Duggan, et al, U.S. Pat. No. 5,281,585.

The "E" substituent illustrated in Fragment-2 (--NHC(═NH)--NH₂) ismerely illustrative of one of the various "E" substituents encompassedby the invention. Fragments similar to the structure of Fragment-2,where E is --NH₂, --SC(═NH)--NH₂ or --C(═NH)--NH₂, can easily besynthesized by techniques that are well known in the art.

The "Y" substituent illustrated in Fragment-2 is --CO-G, where G is:##STR32## where: J is --S-- and L is ##STR33## and R⁹ and R¹⁰ are H.Fragments similar to the structure of Fragment-2, where G is: ##STR34##and J is --SO-- or --SO₂ -- can easily be synthesized by techniques thatare well known in the art. Fragments where J is --O-- can be synthesizedby techniques illustrated in J. Am. Chem. Soc. 114: 1854-1863 (1992), J.Med. Chem. 38:76-85 (1995), and J. Med. Chem. 37:3492-3502 (1994).Lastly, fragments where J is --NR⁶ --, where R⁶ is H, C₁₋₆ alkyl orbenzyl, can be synthesized by techniques illustrated in J. Med. Chem.37:3492-3502 (1994). All of these references are incorporated herein byreference.

Fragments similar to the structure of Fragment-2, where G is: ##STR35##and L is: ##STR36## where R⁷, R⁸, R⁹ and R¹⁰ are not H, can also beeasily synthesized by techniques that are well known in the art.

Fragments similar to the structure of Fragment-2, where G is H, COOR¹¹,CONR¹¹ R¹², --CF₃ or --CF₂ CF₃, can easily be synthesized by techniquesthat are well known in the art.

Fragments similar to the structure of Fragment-2, where G is: ##STR37##and U and V are the various substituents (--O--, --S--, --N--, --N(H)--)can also easily be synthesized by well known techniques illustrated inJ. Med. Chem. 38: 1355-1371 (1995) and J. Med. Chem. 37:2421-2436(1994).

Fragments similar to the structure of Fragment-2, where "Y" is aboron-containing group can easily be synthesized by techniques that arewell known in the art. See for example, J. Org. Chem. 60:3717-3722(1995).

The mechanisms for coupling the various fragments used to synthesize thecompounds of the instant invention, are also well known in the art.

Without further elaboration, it is believed that one skilled in the artcan utilized the present invention to its fullest extent. Therefore, thefollowing preferred specific embodiments are to be construed as merelyillustrative and do not limit the remainder of the disclosure in any waywhatsoever.

EXAMPLE 1 ##STR38##

To a suspension of Boc-Arg(Tos)--OH (2 g, 4.7 mmol) in DMF (20 mL) at 0°C. is added MeNHOMeHCl (1 g, 10.3 mmol), DIEA (6 mL) and BOP (2.5 g, 5.6mmol). The solution is stirred at 0° C. for 10 hours. DMF is evaporatedby vacuum. The oily residue is dissolved in EtOAc (200 mL) and water (20mL). The organic layer is washed with sat. NaHCO₃, water (20 mL), 1 MHCl (10 mL) and sat. NaCl (2×20 mL). The organic layer is dried overMgSO₄, filtered and evaporated to give a suspension. The suspension isfiltered, washed with cold EtOAc (10 mL) and dried to giveBoc-Arg(Tos)-N(Me)OMe, shown above, (1.5 g, 70 % yield). FAB-MS(M+H)+=472

EXAMPLE 2 ##STR39##

To a solution of thiazole (2.5 g, 29 mmol) in THF (25 mL) at -78° C. isadded n-BuLi (1.6 M in hexane, 19 mL) dropwise. The mixture is stirredfor 30 minutes. Then a solution of Boc-Arg(Tos)-N(Me)OMe, from Example1, (1.7 g, 3.6 mmol) in THF (50 mL) is added to the lithiothiazolemixture at -78° C. The solution is stirred for 2 hours. 1M HCl (30 mL)is added to the reaction mixture and warmed to room temperature. Themixture is extracted with EtOAc (100 mL). The organic layer is washedwith sat. NaCl (30 mL), dried over MgSO₄, filtered and evaporated. Thecrude oily residue is purified by flash column over SiO₂ (50% EtOAc inCH₂ Cl₂) to give Boc-Arg(Tos)-Thiazole, shown above, (1.5 g, 84% yield)as a powder. DCI -MS (M+H)+=496

EXAMPLE 3 ##STR40##

To a solution of Boc-Arg(Tos)-Thiazole, from Example 2, (300 mg, 0.6mmol) in CH₂ Cl₂ (10 mL) at 0° C. is added TFA (10 mL). The solution isstirred at 0° C. for 2 hours. The solvent and excess TFA are evaporatedto an oily residue which is used directly without further purification.

EXAMPLE 4 ##STR41##

This compound was prepared according to the procedures described in U.S.Pat. No. 5,281,585 as a colorless foam.

EXAMPLE 5 ##STR42##

The compounds of Example 4 (1.6 g, 5 mmol) and Example 3 (5 mmol) aredissolved in DMF (5 mL) and cooled to 0° C. The solution is neutralizedwith DIEA (3 mL) followed by the addition of coupling reagent BOP (2.7g, 6 mmol). The solution is stirred for 1-2 h and HPLC analysis showsthe completion of reaction. Solvent is removed by vacuum at 30° C. Theresidue is dissolved in a mixture of EtOAc-H₂ O (50 mL:10 mL) andaqueous layer is discarded. The organic layer is washed with sat. NaHCO₃(2×10 mL), sat. NaCl (2×10 mL), dried over MgSO₄, filtered andevaporated. The residue is purified by RP-HPLC to give the compoundshown above, as a powder.

EXAMPLE 6 ##STR43##

100 mg of the compound of Example 5, 1 ml of anisole and 4 drops ofMeSEt are placed in HF-cleavage vessel and cooled under liquid N₂. 10 mlof HF is then condensed and the mixture is stirred at 0° C. for 1.25 h.HF is removed under vacuum to give a gum-like residue. The residue istriturated with 20 ml of 50% Et₂ O-hexane and the solvent removed byfiltration. The gum residue is dissolved in 10 mL of 0.1% TFA in waterand 1 mL of 0.1% TFA in MeCN and purified by RP-HPLC to give thecompound shown above, as a powder.

EXAMPLE 7 Determination of IC₅₀

The compounds of the present invention are first dissolved in a bufferto give solutions containing concentrations such that assayconcentrations range from 0-100 μM. In assays for thrombin,prothrombinase and factor Xa, a synthetic chromogenic substrate would beadded to a solution containing a test compound and the enzyme ofinterest and the residual catalytic activity of that enzyme would thenbe determined spectrophotometrically.

The IC₅₀ of a compound is determined from the substrate turnover. TheIC₅₀ is the concentration of test compound giving 50% inhibition of thesubstrate turnover. Preferred compounds of the invention desirably havean IC₅₀ of less than 500 nM in the factor Xa assay, preferably less than200 nM, and more preferably less than 100 mM. Preferred compounds of theinvention desirably have an IC₅₀ of less than 4.0 μM in theprothrombinase assay, preferably less than 200 nM, and more preferablyless than 10 nM. Preferred compounds of the invention desirably have anIC₅₀ of greater than 1.0 μM in the thrombin assay, preferably greaterthan 10.0 μM, and more preferably greater than 100.0 μM.

Amidolytic Assays for determining protease inhibition activity

Factor Xa and thrombin assays are performed at room temperature, in 0.02M Tris HCl buffer, pH 7.5, containing 0.15 M NaCl. The rates ofhydrolysis of the para-nitroanilide substrate S-2765 (Chromogenix) forfactor Xa, and the substrate Chromozym TH (Boehringer Mannheim) forthrombin following preincubation of the enzyme with the test compoundfor 5 minutes at room temperature are determined using a Softmax 96-wellplate reader (Molecular Devices), monitored at 405 nm to measure thetime dependent appearance of p-nitroanilide.

The prothrombinase inhibition assay is performed in a plasma free systemwith modifications to the method as described by Sinha, et al., Thromb.Res., 75:427-436 (1994). The activity of the prothrombinase complex isdetermined by measuring the time course of thrombin generation using thep-nitroanilide substrate Chromozym TH. The assay consists of a 5 minutepreincubation of selected compounds to be tested as inhibitors with thecomplex formed from factor Xa (0.5 nM), factor Va (2 nM), phosphatidylserine:phosphatidyl choline (25:75, 20 μM) in 20 mM Tris HCl buffer, pH7.5, containing 0.15 M NaCl, 5 mM CaCl₂ and 0.1% bovine serum albumin.Aliquots from the complex-test compound mixture are added to prothrombin(1 nM) and Chromozym TH (0.1 mM). The rate of substrate cleavage ismonitored at 405 nm for two minutes. Several concentrations of a giventest compound are assayed in duplicate. A standard curve of thrombingeneration by an equivalent amount of untreated complex is then used fordetermination of percent inhibition.

EXAMPLE 8

The antithrombotic efficacy of the compounds of this invention canreadily be evaluated using a series of studies in rabbits, as describedbelow. These studies are also useful in evaluating a compounds effectson hemostasis and its the hematological parameters.

Antithrombotic Efficacy in a Rabbit Model of Venous Thrombosis

A rabbit deep vein thrombosis model as described by Hollenbach, et al.,Thromb. Haemost. 71:357-362 (1994), is used to determine the in vivoantithrombotic activity of the compounds of the present invention.Rabbits are anesthetized with I.M. injections of Ketamine, Xylazine, andAcepromazine cocktail.

A standardized protocol consists of insertion of a thrombogenic cottonthread and copper wire apparatus into the abdominal vena cava of theanesthetized rabbit. A non-occlusive thrombus is allowed to develop inthe central venous circulation and inhibition of thrombus growth is thenused as a measure of the antithrombotic activity of the compound beingevaluated. Test agents or control saline are administered through amarginal ear vein catheter. A femoral vein catheter is used for bloodsampling prior to and during steady state infusion of the compound beingevaluated. Initiation of thrombus formation will begin immediately afteradvancement of the cotton thread apparatus into the central venouscirculation. The compounds being evaluated are administered from time=30minutes to time=150 minutes at which point the experiment is terminated.The rabbits are euthanized and the thrombus excised by surgicaldissection and characterized by weight and histology. Blood samples arethen analyzed for changes in hematological and coagulation parameters.

Although the invention has been described with reference to thedisclosed embodiments, those skilled in the art will readily appreciatethat the specific experiments detailed are only illustrative of theinvention. It should be understood that various modifications can bemade without departing from the spirit of the invention. Accordingly,the invention is limited only by the following claims.

What is claimed is:
 1. A compound having the formula: ##STR44## whereinR² is H, C₁₋₆ alkyl, C₃₋₆ cycloalkyl, C₁₋₃ alkylaryl, C₁₋₃ alkyl-C₃₋₈cycloalkyl or aryl;R³ is H, C₁₋₆ alkyl, or R² and R³ are taken togetherto form a carbocyclic ring; R¹³ is H, C₁₋₃ alkyl, C₁₋₃ alkylaryl, aryl,or --CF₃ ; R¹ is H, C₁₋₃ alkyl, C₁₋₃ alkylaryl, aryl, or --CF₃ ; R¹⁴ isH, C₁₋₃ alkyl, C₁₋₃ alkylaryl, aryl, or --CF₃ ; m is an integer from0-3; n is an integer from 0-6; p is an integer from 0-4; s is an integerfrom 0-2; W is C₁₋₆ alkyl, C₃₋₈ cycloalkyl, C₁₋₆ alkenyl, or aryl; K isselected from the group consisting of a direct link, C₃₋₈ cycloalkyl,aryl; E is selected from the group consisting of R²⁶, --NR²⁶ R²⁷,##STR45## R²⁶, R²⁷, R²⁸ and R²⁹ are independently selected from thegroup consisting of H, --OH, C₁₋₆ alkyl, aryl and C₁₋₄ alkylaryl; R³⁰ isselected from the group consisting of H, C₁₋₆ alkyl, aryl and C₁₋₄alkylaryl; and R³¹ is selected from the group consisting of H, C₁₋₆alkyl, aryl and C₁₋₄ alkylaryl, or all optical isomers thereof.
 2. Thecompound of claim 1 where R₂ is H or C₁₋₆ alkyl.
 3. The compound ofclaim 1 where R³ is H.
 4. The compound of claim 1 where R¹³ is H, C₁₋₃alkyl or --CF₃.
 5. The compound of claim 1 where R₁ is H, C₁₋₃ alkyl or--CF₃.
 6. The compound of claim 1 where R¹⁴ is H, C₁₋₃ alkyl or --CF₃.7. The compound of claim 1 where m is an integer from 0-1.
 8. Thecompound of claim 1 where n is an integer from 1-4.
 9. The compound ofclaim 1 where p is
 3. 10. The compound of claim 1 where s is
 0. 11. Thecompound of claim 1 where W is C₁₋₄ alkyl, C₅₋₆ cycloalkyl or aryl. 12.The compound of claim 1 where K is a direct link.
 13. The compound ofclaim 1 where E is --NH₂, --NHC(═NH)--NH₂ or --SC(═NH)--NH₂.
 14. Acompound of claim 1, having the formula: ##STR46##
 15. A compound ofclaim 14, having the formula:
 16. A compound of claim 14, wherein R¹⁴ isH, C₁₋₃ alkyl or --CF₃.
 17. A compound of claim 14, wherein R¹⁴ is H,C₁₋₃ alkyl or --CF₃.
 18. A compound of claim 14, wherein W is C₁₋₄alkyl, C₅₋₆ cycloalkyl, or aryl.
 19. A compound of claim 14, having thefollowing stereochemical formula:
 20. A compound of claim 19, having thefollowing stereochemical formula:
 21. A compound of claim 19, having thefollowing stereochemical formula:
 22. A compound of claim 19 having thefollowing stereochemical formula:
 23. A compound of claim 19, having thefollowing sterochemical formula;
 24. A pharmaceutical composition forpreventing or treating a condition in a mammal characterized byundesired thrombosis comprising a pharmaceutically acceptable carrierand an effective amount of the compound of claim
 1. 25. A pharmaceuticalcomposition for preventing or treating a condition in a mammalcharacterized by undesired thrombosis comprising a pharmaceuticallyacceptable carrier and an effective amount of the compound of claim 14.26. A pharmaceutical composition for preventing or treating a conditionin a mammal characterized by undesired thrombosis comprising apharmaceutically acceptable carrier and an effective amount of thecompound of claim
 19. 27. A pharmaceutical composition for preventing ortreating a condition in a mammal characterized by undesired thrombosiscomprising a pharmaceutically acceptable carrier and an effective amountof the compound of claim
 20. 28. A pharmaceutical composition forpreventing or treating a condition in a mammal characterized byundesired thrombosis comprising a pharmaceutically acceptable carrierand an effective amount of the compound of claim
 21. 29. Apharmaceutical composition for preventing or treating a condition in amammal characterized by undesired thrombosis comprising apharmaceutically acceptable carrier and an effective amount of thecompound of claim
 22. 30. A pharmaceutical composition for preventing ortreating a condition in a mammal characterized by undesired thrombosiscomprising a pharmaceutically acceptable carrier and an effective amountof the compound of claim 23.